Funded in December, 2005: $200000 for 3 years

How do the millions of brain cells establish billions of connections (i.e., synapses) with one another? How do they find and choose suitable partners with which to make synapses in the vastness of the immature brain? Can errors in synapse formation lead to mental retardation or autism? We want to answer these questions using state-of-the-art imaging techniques to visualize synaptogenesis and pharmacology to investigate the mechanisms involved at the level of single synapses.

As neurons grow and mature during brain development, they extend two sets of cellular processes: dendrites (recipients of electrical signals from other neurons) and axons (cables that propagate the signals further). In the adult brain, dendrites of excitatory neurons are covered with tiny lollipop-shaped appendages known as dendritic spines. Sitting across the synapse from axons, spines contain the receptors for glutamate, a neurotransmitter released by axon terminals. The exact function of spines is not known, but evidence suggests they are important for learning and memory. Indeed, their size and shape can change depending on neural activity and sensory experience.

Before spines appear, developing dendrites are first covered with longer and thinner protrusions called filopodia. We have shown that filopodia are motile, appearing and disappearing over time scales of seconds. This suggests an exploratory role for filopodia and has led to the proposal that they may be important for finding axons and establishing early synapses. Moreover, because filopodia are precursors of spines, it has been argued that filopodia may transform into spines. We also found that glutamate causes filopodia to grow longer. This fits well with a model in which glutamate released by axons near a given dendrite may help recruit filopodia to establish early synapses. Filopodia that make successful synapses may later stabilize and give rise to spines. It is conceivable that defects in any of the steps that lead from the dynamic behavior of filopodia to spine elaboration could lead to dysfunctional neural circuits that predispose individuals to autism and mental retardation.

Interestingly, autopsy studies have shown that individuals with various forms of mental retardation and/or autism have abnormal dendritic spines. In particular, the only microscopic brain anomaly found in fragile X syndrome, the most common inherited form of mental retardation and autism, is the presence of thin and tortuous spines that resemble filopodia. We want to test the hypothesis that fragile X is caused by a defect in filopodia, which impairs their ability to mature into spines. Recent studies using a genetic mouse model of fragile X have confirmed that brains of these mutant mice have the same aberrant spines. But no one has yet examined the kinetics of filopodia and their role in spine generation in these mice, during the early mouse brain development.

We are interested in learning more about the process of spine elaboration during normal development and disease. We intend to use sophisticated laser microscopy techniques to image dendritic filopodia in the intact brain of living mice. We will implant small optical "windows" onto the skulls of newborn mice so we can collect images of individual filopodia and spines. By comparing normal mice to fragile X mutant mice, we will be able to identify potential abnormalities of filopodia that disrupt their dynamics or interfere with their capacity to respond to glutamate,or their ability to make spines. Our experiments are designed to discover new molecular targets that can be exploited for therapeutic purposes in fragile X and other neuropsychiatric disorders characterized by aberrant neural circuits.

Our goal is to identify new molecular pathways that can be exploited for therapeutic purposes in neurodevelopmental disorders characterized by autism and mental retardation. Fragile X syndrome is the most common inherited form of mental retardation and autism. The vast majority of affected individuals have an expanded CGG repeat within the Fmr1 gene, leading to a loss of function phenotype. How this translates into the complex neuropsychiatric phenotype of fragile X is not yet understood.

Interestingly, brains of children with fragile X have abnormally long and densely packed dendritic spines, and the same defect has also been observed in Fmr1 knockout mice. These bizarre spines are reminiscent of dendritic protrusions normally present in the developing brain: filopodia. It has therefore been proposed that fragile X may be caused by a failure in the normal transition from filopodia to spines. Yet, the development of dendritic filopodia in the first postnatal days, at the peak of their expression, has never been examined in fragile X mice.

We recently described the developmental maturation of dendritic filopodia in Layer 5 pyramidal neurons in acute slices of mouse primary visual cortex. We established that filopodia in dendritic shafts elongate in response to the neurotransmitter glutamate. This observation suggests that glutamate released by nearby axons may recruit filopodia to form early synaptic contacts. These filopodial synapses presumably stabilize filopodia and allow them to mature into spines. It is conceivable that impaired filopodial dynamics that interfere with their ability to make synapses, might lead to defects in spine elaboration similar to those seen in fragile X. The notion that glutamate neurotransmission might be perturbed in fragile X is further supported by a recent theory about fragile X known as the metabotropic glutamate receptor hypothesis.

We therefore want to test the hypothesis that a defect in filopodia, linked to abnormal glutamate signaling or downstream molecular pathways, impairs their ability to mature into spines, leading to the dendritic abnormalities found in fragile X. Because filopodia are motile, exploring this would require imaging neurons in real time, preferably with preserved connectivity. Therefore, the experiments should be done in vivo, because the disruption of circuits that occurs after preparing acute or cultured slices is likely to have an impact on these phenomena. Sadly, it has not yet been possible to image neurons in the intact brain of living newborn mice.

To overcome this challenge, we have developed a novel and sophisticated method for imaging filopodia in neonatal mice using two-photon microscopy. We intend to use this state-of-the-art brain imaging technique to examine the maturation of dendritic filopodia and their role in synapse and spine formation during normal development and in fragile X mice. First, we will look for abnormalities in filopodia dynamics in the mutant mice, using two-photon microscopy through a small optical 'window' in the skull. Second, we will examine whether the ability of filopodia to respond to glutamate is impaired in the mutant mice, using two-photon glutamate uncaging together with pharmacology to interfere with various signaling pathways. Third, we will test whether the filopodia to spine transition is affected in fragile X.

The experiments proposed combine the use of innovative imaging techniques, pharmacology, and molecular approaches to unravel the defective signaling pathways that lead to aberrant spine maturation in fragile X. This research may uncover new strategies for treating autism.

Follow-on Funding:
Dr. Portera-Cailliau's laboratory has been funded by the Fragile X Research Foundation (FRAXA). Grants are also pending from the NIH (RO1) and other private foundations, including the March of Dimes Foundation.

Hypothesis:We want to test the hypothesis that a defect in dendritic filopodia, linked to abnormal glutamate signaling or downstream molecular pathways, impairs their ability to mature into spines, leading to the dendritic abnormalities found in fragile X syndrome.

Goals:We want to identify new molecular pathways that can be exploited for therapeutic purposes in neurodevelopmental disorders characterized by autism and mental retardation, including fragile X syndrome (FXS). Brains of individuals with FXS have abnormally long and densely packed dendritic spines, and the same defect has also been observed in a knockout mouse model of FXS. These bizarre spines are reminiscent of dendritic protrusions normally present in the developing brain: filopodia. Yet, it is not known whether abnormalities occur in filopodia of fragile X mice in the first postnatal days, at the peak of their expression.We intend to use state-of-the-art brain imaging techniques to examine the maturation of dendritic filopodia and their role in spine formation in fragile X mice. First, we will look for abnormalities in filopodia dynamics in the mutant mice. We have shown that filopodia elongate when glutamate is applied to dendrites and proposed that this is how axons recruit filopodia to make early synapses. In the second set of experiments, we will explore the role of mGluRs and Rho GTPases in glutamate-mediated growth of filopodia.

Methods:
We have developed a novel and sophisticated method for imaging filopodia in neonatal mice using two-photon microscopy through a small optical '"indow" in the skull. Two-photon glutamate uncaging will be used to examine the mechanisms of glutamate-induced filopodia growth. We will also use pharmacological and molecular approaches to interfere with the Rho GTPase signaling pathway.

Amit Etkin is investigating
the use of repetitive transcranial magnetic stimulation in combination with
whole-brain EEG and functional MRI to treat depression and help unravel its
underlying brain circuitry.